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The FET family of atypical RNA-binding proteins includes Fused in sarcoma (Fus), Ewings sarcoma (EWS), and the TATA-binding protein-associate factor 15 (TAF15). All FET family members are highly conserved from fish to mammals, suggesting an independent and specialized requirement for each protein. Fus is necessary for the proper splicing of genes required for mesoderm differentiation and cell adhesion in Xenopus, but the role, if any, that EWS and TAF15 play in development remains unknown. Here we define the role maternally deposited and zygotically transcribed TAF15 plays in development. We find that TAF15 is essential for the proper development of dorsoanterial neural tissues, and by sequencing the RNA from single TAF15-depleted embryos and measuring changes in transcript abundance and exon usage we found TAF15 regulates dorsoanterior neural tissue development through regulating fgfr4 and ventx2.1. Intriguingly, we find that TAF15 uses two distinct mechanisms to downregulate FGFR4 expression: 1) retention of a single intron within fgfr4 and 2) reduction of total fgfr4 transcript. Intron retention was identified when both maternal and zygotic TAF15 is depleted, while depletion of zygotic TAF15 alone leads to regulation of fgfr4 total transcripts. In this study we find that TAF15 plays an integral and pleiotropic role in the development of dorsoanterior neural tissues and further identify two novel mechanisms of gene regulation by TAF15, suggesting TAF15 gene regulation is target and cofactor-dependent, subject to the milieu of factors that are present at different times of development.

Significance The majority of transcription generates noncoding RNAs, most of which are uncharacterized. Using RNA-seq on cultured mouse sclerotome, we identified PEAT , a long-noncoding RNA (lncRNA) adjacent to a key regulator of sclerotome, Pax1 . We deleted the entire PEAT -transcribed unit using CRISPR/Cas9 and analyzed RNA-seq from mutant embryos. While some lncRNAs regulate the expression of their proximal genes, our analysis showed Pax1 expression to be unchanged. However, we identified 60 ribosomal proteins with elevated expression, and found evidence that bone morphogenetic protein signaling is slightly elevated in PEAT mutants. This study reveals a role for the lncRNA PEAT in sclerotome development and shows next-generation sequencing to be a powerful tool to reveal surprising functions for lncRNAs.

SummaryAlternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping), 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Here we investigated the function of the atypical RNA-binding protein fus/TLS (fused in sarcoma/translocated in sarcoma) during early frog development. We found that fus is necessary for proper mRNA splicing of a set of developmental regulatory genes during early frog development and gastrulation. Upon fus knockdown, embryos fail to gastrulate and show mesodermal differentiation defects that we connect to intron retention in fgf8 (fibroblast growth factor 8) and fgfr2 (fgf receptor 2) transcripts. During gastrulation, the animal and marginal regions dissociate, and we show that this is caused, at least in part, by intron retention in cdh1 transcripts. We confirm the specificity of splicing defects at a genomic level using analysis of RNA sequencing (RNA-seq) and show that 3%–5% of all transcripts display intron retention throughout the pre-mRNA. By analyzing gene ontology slim annotations, we show that the affected genes are enriched for developmental regulators and therefore represent a biologically coherent set of targets for fus regulation in embryogenesis. This shows that fus is central to embryogenesis and may provide information on its function in neurodegenerative disease.

We have performed a high-capacity, semiquantitative, reverse transcriptase-polymerase chain reaction screen for expression of fibroblast growth factor (FGF) and transforming growth factor beta (TGFbeta) family genes as well as their cognate receptors. By using cDNA prepared from embryonic day 12 to postnatal day 0 embryonic mouse pancreas, we have identified several factors potentially involved in the development of the endocrine pancreas. We find high-level early expression of TGFbeta-1 and -2, and constitutive expression of TGFbeta-3 and their receptors. Of the Inhibin/Activin members, we found exclusively Inhibin-alpha and Activin-betaB to be expressed, and the BMP family was represented by BMP4, BMP5, and BMP7. The predominant forms of the BMP and Activin type II receptors were ActR-IIB and BMPR-II and of the type I receptors, BMPR-1A and -1B were the highest expressed. FGF1, FGF7, FGF9, FGF10, FGF11, and FGF18 were also expressed in the pancreas at varying time points and levels, as well as FGF receptor forms FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, and FGFR4. To gain insight into the biological function, we misexpressed members of these families in the pancreas by using the early pancreas promoter Pdx1. Misexpression of FGF4 results in disruption of the pancreas morphology with epithelial structures interspersed in stroma tissue. The endocrine compartment was reduced to scattered single cells, and the exocrine consisted of unbranched ductal epithelia with acinar structures budding off. In contrast, misexpression of BMP-6 resulted in complete agenesis of the pancreas and reduced the size of the stomach and spleen dramatically and caused fusion of the liver and duodenum.

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic α cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr −/− ). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr −/− mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to α cell, and to a lesser extent, δ cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1–37) and GLP-1 amide (1–36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr −/− mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and α and δ cell numbers. Furthermore, the lean phenotype of Gcgr −/− mice suggests glucagon action may be involved in the regulation of whole body composition.

Neuronal subtypes originate from an undifferentiated neural epithelium that is progressively divided into progenitor domains by homeodomain transcription factors such as members of the Nkx family. Here we report the functional analysis of Nkx6.1 and Nkx6.2 in Xenopus. While Nkx6.2 is expressed early in a large region of the medial neural plate, Nkx6.1 is restricted to a region overlapping with the region of motor neuron formation. By mRNA injection we show that both can inhibit primary neurogenesis as well as expression of intermediate neural plate markers. However, they do not form auto-regulatory loops and fail to induce ectopic motor neurons as they do in the chick. Using morpholino-mediated knockdown in Xenopus laevis and Xenopus tropicalis we show that Nkx6.1 knockdown results in paralyzed tadpoles. Using DiI labeling and immunohistochemistry we show that the underlying mechanism is a failure of spinal motor neurons to extend axons to their targets. Analysis of neural pattern reveals that ventral Lhx3+ and Pax2+ interneurons are dependent on Nkx6.1 function, but overall neural patterning is not. This study illustrates that while important aspects of Nkx6 gene function are conserved in vertebrate neural patterning, others are not.

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633 , see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes.

We have performed an expression cloning screen in Xenopus laevis with the aim of isolating novel gene activities from the neural plate. Of 8,064 clones screened, we isolated 61 clones that affected either neural plate patterning or tadpole morphology. Of these, 20 clones encoded RNA binding proteins, and the majority of these are heterogeneous nuclear ribonucleoproteins (hnRNPs) or SR-proteins, which are associated with alternative splicing. All of these genes are expressed in the nervous system, and in several cases specific to neural tissue. Injecting mRNA encoding these proteins results in neural plate mispatterning and abnormal muscle segmentation. To initiate characterization of these proteins, we selected Rbmx as a candidate for deeper analysis. Using morpholino mediated knockdown, we show that Rbmx is necessary for normal anterior neural plate patterning, neurogenesis, neural crest development, and muscle segmentation. Developmental Dynamics 237:1755–1766, 2008. © 2008 Wiley-Liss, Inc.

Six distinct fibroblast growth factors (FGF5) have been detected in pancreatic islets by immunohistochemistry (IHC) using commercially available antisera. We show here that these antisera are useful for Western blotting but that only two are suited for IHC. By Western blotting, these antisera detect recombinant FGFs. Detection can be eliminated by preabsorption with immunizing peptide but not with irrelevant peptide. By IHC we find specific labeling of islets with anti-FGF1 and anti-FGF2 antisera. Labeling can be abolished by preabsorption with the immunizing peptides. In contrast, prominent staining of islets by anti-FGF4, −FGF5, −FGF7, and −FGF10 antisera is unspecific because the staining cannot be competed by preabsorption with the immunizing peptides.

It is well established that gut and pancreas development depend on epithelial-mesenchymal interactions. We show here that several Wnt, Frizzled, and secreted frizzled-related protein (sFRP) encoding mRNAs are present during mouse pancreatic morphogenesis. Wnt5a and 7b mRNA is broadly expressed in foregut mesenchyme starting around embryonic day 10 in mice. Other members expressed are Wnt2b, Wnt5b, and Wnt11. In addition, genes for the Wnt receptors, Frizzled2, 3, 4, 5, 6, 7, 8, and 9 are expressed. To understand potential Wnt functions in pancreas and foregut development in vivo, we analyzed transgenic F0 mouse fetuses expressing Wnt1 and 5a cDNAs under control of the PDX-1 gene promoter. In PDX-Wnt1 fetuses, the foregut region normally comprising the proximal duodenum instead resembles a posterior extension of the stomach, often associated with complete pancreatic and splenic agenesis. Furthermore, the boundary between expression domains of gastric and duodenal markers is shifted in a posterior direction. In PDX-Wnt5a fetuses, several structures derived from the proximal foregut are reduced in size, including the pancreas, spleen, and stomach, without any apparent shift in the stomach to duodenum transition. In these fetuses, overall pancreatic morphology is changed and the pancreatic epithelium is dense and compact, consistent with Wnt5A effects on cell movements and/or attachment. Taken together, these results suggest that Wnt genes participate in epithelial-mesenchymal signaling and may specify region identity in the anterior foregut. © 2002 Wiley-Liss, Inc.